This invention relates to sterilization and more specifically to sterilization processes for use with biological materials, such as organ replacements and homografts, which processes exhibit efficacy against difficult-to-kill bacteria and bacterial spores wherein the effect upon the physical properties of the biological material being sterilized is controllable.
Sterilization techniques are widely used and important in industries such as food processing and health care. Saturated steam at temperatures above 110xc2x0 C. has frequently been used to destroy microorganisms, such as microbial spores. Certain articles, particularly those used for health care which consist of or incorporate biological tissue, cannot withstand the temperatures and moisture of steam sterilization, and oftentimes such articles are also considered not to be suitable for sterilization by ionizing radiation. Gaseous sterilants have been developed which function at relatively low temperatures and thus may offer an attractive alternative. Ethylene oxide is a commonly used gaseous sterilant which is often used for medical product sterilization; however, in certain instances, the presence of residual ethylene oxide is considered to be detrimental, even in small quantities. Allografts and other implants containing biological tissue have been sterilized by immersion in antibiotic mixtures, but such processes are very expensive and do not destroy certain bacterial spores and viruses. Accordingly, improved methods of sterilization for medical products that include biological tissue have continued to be sought which would not otherwise significantly alter the physical characteristics of the medical products, particularly allografts and xenografts for which the physical effect upon the medical products should be closely controlled.
Our U.S. Pat. No. 5,911,951 describes an excellent sterilization process for biological tissue by treating with a water-soluble carbodiimide, such as EDC, in the presence of a lower alkanol, such as isopropanol.
It has now been found that medical products which include biological tissue, e.g. allografts and xenografts is such as replacement organs, ligaments, tendons, vascular grafts and the like, as well as acellular material including items made of extracted collagen or using a process such as that taught in U.S. Pat. No. 4,776,853, can be effectively sterilized by treatment with a bactericidal coupling agent of the type known to create amide linkages between amines and carboxyl moieties in a manner so that there is either no significant change or a closely controllable change in the physical character of the biological material being sterilized. Sterilization treatment is preferably carried out at a temperature above ambient in a buffered solution, that may optionally contain isopropyl alcohol or an equivalent alcohol in an amount effective to promote penetration of said coupling agent into the cells of the microorganisms. The sterilization treatment is carried out in the presence of protecting agents that complex with and protect either residual amine groups or residual carboxyl groups on the proteinaceous biological material that is being treated, thereby carefully controlling the amount of cross-linking thereof so as not to undesirably change the physical characteristics of the resultant biological material. The residuals from such treatment are nontoxic, biocompatible, and water-soluble, so that they can easily be washed off the tissue before packaging or implantation in a human body.
It was surprising to find that biological tissue can be effectively sterilized using coupling agents that create amide bonds, while at the same time either avoiding the occurrence of any significant cross-linking within the biological material itself or alternatively carefully controlling the amount of cross-linking which occurs.
In one particular manner, the invention provides a process for sterilization of biological tissue material, which process comprises treating such material with an aqueous solution containing (a) an amount of a water-soluble coupling agent capable of creating amide bonds that is effective to achieve sterilization and (b) an amount of a protecting agent which is effective to complex with potentially reactive amine or carboxyl moieties on the biological tissue material so as to deter such complexed moieties from taking part in an amide-forming cross-linking reaction and thereby limit the amount of cross-linking, and maintaining such treatment for a time and at a temperature which is sufficient to achieve penetration of said coupling agent into the cells of microorganisms carried by such material and effectively kill such microorganisms.
In another particular manner, the invention provides a process for sterilization of biological tissue, which process comprises treating such material with a protecting agent which is effective to complex with potentially reactive amine or carboxyl moieties on the biological tissue material so as to deter such complexed moieties from taking part in an amide-forming cross-linking reaction and thereby limit the amount of cross-linking, treating such material with a water-soluble coupling agent capable of creating amide bonds that is effective to achieve sterilization, and maintaining such treatment with said coupling agent for a time and at a temperature which is sufficient to achieve penetration of said coupling agent into the cells of microorganisms carried by such material and effectively kill such microorganisms.
In a further particular manner, the invention provides a process of sterilization of biological tissue that has been rendered acellular either before or after cross-linking or that has not been cross-linked.
Medical products which consist of or incorporate biological tissue require sterilization prior to packaging or prior to implantation into a patient, and for certain products, the sterilant should be one that leaves no undesirable residue and that either does not change the physical characteristics of the medical product or only changes it in a carefully controllable manner. For example, for allografts, changes in the structure or the rigidity of the biological tissue are considered detrimental and to be avoided. Allografts, sometimes referred to as homografts, include replacement organs and connective tissues, e.g. cartilage, tendons, ligaments, bone, and muscle tissue, and are generally desirably transplanted into a receiving patient without change in structure or character. On the other hand, certain medical products which include animal tissue, such as prosthetic tissue valves for heart valve replacement, e.g. porcine aortic valves or tissue valves constructed from bovine pericardium, may have been previously fixed, i.e. cross-linked, to carefully provide them with a character and flexibility desired for long term operation. Accordingly, any change to such carefully tailored physical characteristics, as by rigidifying the tissue, may be considered to constitute an undesirable alteration of the product. Still other tissue-engineered products may desirably have a variable duration of resorption in instances where it is desired that the body remodel the product following implantation.
In the foregoing context, once it was discovered that amide-creating coupling agents could be effectively used to sterilize articles by completely inactivating microorganisms and spores, it was decided to investigate if such a sterilization treatment could be adapted to sterilizing allografts and other such engineered tissue products that include mammalian biological tissue. It was surprisingly found that the potentially accompanying undesirable cross-linking of the proteinaceous biological tissue could be minimized or carefully controlled by carrying out the sterilization treatment in the presence of protective agents that would temporarily or permanently complex with the potentially reactive amine or carboxyl moieties on the proteinaceous material and prevent extensive cross-linking that would otherwise change the physical properties of the biological material, as by rigidifying it.
The term xe2x80x9ccoupling agentxe2x80x9d is herein used to refer to a chemical reagent that facilitates the formation of amide bonds. Such bonds may be formed between reactive amines and reactive carboxyls on enzymes or proteins; they may also be formed with and between the reactive carboxyl or amine moieties located on and within bioprosthetic tissue as taught in U.S. Pat. No. 5,733,339, the disclosure of which is incorporated herein by reference. Those having skill in peptide synthesis and related arts will be familiar with such reagents, e.g. water-soluble carbodiimides; a list of such coupling agents is found in the book: Bioconjugate Techniques by Greg T. Hermanson published by Academic Press 1996. This sterilization process which destroys bacteria and spores by treatment with such coupling agents is promoted when carried out in the presence of a C2 to C4 alkanol. For purpose of this application, sterilization is considered to be achieved when the conditions of treatment achieve at least a log 6 reduction in bacteria content, or would so reduce a standard bacteria sample.
A water-soluble coupling agent is preferably chosen so the treatment of biological tissue materials can be effected in aqueous solution. The preferred coupling agent is 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydro-chloride (EDC); alternative suitable coupling agents include other water-soluble carbodiimides such as 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, N,Nxe2x80x2-carbonyldiimidazole, Woodward""s Reagent K and mixtures of such carbodiimides.
Concentrations of the coupling agent can be varied within certain limits, and lower concentrations may be effectively employed if the coupling agent is used in combination with a lower alkanol. When used without an accompanying lower alkanol, the coupling agent is preferably used in a concentration between about 25 millimolar (mM) and about 150 mM; however, often concentrations between about 35 mM and about 70 mM are employed, which are considered effective to destroy all commonly encountered bacteria and spores at temperatures of between about 35xc2x0 C. and 55xc2x0 C. Duration of sterilization treatment is usually for at least about 6 hours and preferably at least about 12 hours; however, treatment for 24 hours is common.
The solution may optionally contain up to about 30 volume % of a C2 to C4 alkanol, e.g. ethanol or isopropanol, or an equivalent alcohol; by volume % is meant volume of alcohol relative to volume of solution. Such lower alkanol is believed to assist the coupling agent in effectively penetrating the cell walls of the bacteria, spores or other microorganisms. When an alkanol is used, at least about 10% is used, and between about 15% and about 25% of isopropanol is commonly employed, and the coupling agent may be used at a concentration of only about 5 mM-15 mM although 25 mM may be conveniently used.
The protecting agent may be one which complexes with the reactive amine moieties and/or with the reactive carboxyl moieties that are present on the biological tissue. Advantage has heretofore been taken of the presence of these potentially reactive moieties to stabilize prostheses which include animal tissue, and this stabilization process is commonly referred to as xe2x80x9cfixingxe2x80x9d. Glutaraldehyde has commonly been used as a fixation reagent, and other fixation techniques have employed polyepoxide cross-linking or photo-oxidation. More recently, fixation processes of the types detailed in U.S. Pat. Nos. 5,447,536 and 5,733,339, the disclosures of which are incorporated herein by reference, have been proved to have various advantages. It has now been found that, by complexing protecting agents with these potentially reactive moieties, the amount of cross-linking that occurs in the proteinaceous biological tissue can be minimized or closely controlled, while surprisingly not significantly adversely affecting the bactericidal effect of a sterilization treatment utilizing EDC or an equivalent.
Such a protecting agent can be included in the solution used for the sterilization treatment, or the biological tissue material to be sterilized may be pretreated with the protecting agent. If such pretreatment is used, a slightly lower concentration of the protecting agent may be adequate, inasmuch as there will be greater opportunity for complexing to take place prior to exposure of the proteinaceous material to the coupling agent. Generally, if such pretreatment is employed, the sterilants will then be later added to the solution; however, some blocking agents will link to the tissue so rinsing can take place before sterilizing as mentioned hereinafter.
Potentially reactive amine moieties that are present on the proteinaceous material can be effectively blocked before sterilization with certain protecting agents, such as N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate), acetic anhydride, maleic anhydride and citraconic anhydride, after which rinsing can be effected prior to sterilization. An alternative approach is to use monocarboxylic acids, such as hydroxyproline, acetic acid and propionic acid, as protecting groups that will, in the presence of a coupling agent such as a carbodiimide, preferentially form amide linkages with the active amine moieties, thereby preventing these moieties from forming cross-links with carboxylate groups elsewhere on the proteinaceous material during sterilization. However, when monocarboxylic acids are used as the protecting groups, it may be desirable to employ somewhat greater concentrations of the coupling agent because some of the coupling agent may be diverted from its primary sterilization function.
Instead of complexing the amine moieties, a protecting agent can be chosen to complex the carboxyl moieties. Monoamines which will react with the carboxylic groups can be used, and selection of a hydroxyl monoamine, e.g. ethanolamine or propanolamine, may be preferred. A particularly preferred protecting agent is a combination of ethanolamine and tris(hydroxymethyl)aminomethane (TRIS); TRIS serves two functions because it is also a known buffer that is useful for inclusion in aqueous solutions at physiological pH, although ethanolamine or propanolamine could be used alone as either is capable of serving as both a buffer and a blocking agent. In addition, the hydroxyl groups carried by protecting agents such as TRIS increase the hydrophilicity of the biological tissue and have the effect of rendering it less thrombogenic.
When the protecting agent is included as a part of the aqueous sterilization treatment solution, it is generally employed at a concentration between about 25 mM and about 200 mM, preferably at a concentration between about 50 and about 150 mM, and more preferably at a concentration between about 80 mM and about 120 mM. When pretreatment of the biological tissue material with the protecting agent is employed, the protecting agent, e.g. acetic anhydride or ethanolamine, may generally be used at a concentration between about 5 mM and about 50 mM in an aqueous solution, and preferably at a concentration of at least about 10 mM and, more preferably, at least about 20 mM. When pretreatment is used, it is carried out at least about 5 hours and preferably at least about 12 hours prior to the sterilization treatment because the reagent needs to penetrate the tissue for the chemical reaction to occur.
Reaction conditions for the sterilization process may vary somewhat depending on the specific coupling agent employed. In general, sterilization treatment is carried out in an aqueous buffer solution selected from among those that are well known to those of ordinary skill in this art. Examples of suitable buffers include, but are not limited to, N-2-hydroxyethylpiperazine-Nxe2x80x2-ethanesulfonic acid (HEPES) and 3-(N-morpholino)propanesulfonic acid (MOPS), and the like. As earlier indicated, it is possible to use TRIS as a protecting agent for the potentially reactive carboxylate groups, preferably in combination with ethanolamine, in which case it will serve the dual function of a buffer. Under these circumstances, it may be desirable to use TRIS at a concentration between about 50 and about 100 mM, with about the same concentration of ethanolamine.
The pH and concentration of the buffered solution also may vary, again depending upon the coupling agent employed. All solutions used are preferably filtered through 0.45 xcexcm or smaller filters before use. Preferably, the buffer concentration and pH are chosen to provide an effective sterilization environment while being the least harmful to biological tissue material; this will generally be a physiological pH. For example, with EDC as the coupling agent, the pH of the solution employed is usually between about 6.0 and about 7.0. The temperature of the sterilizing solution is usually maintained between about 25xc2x0 C. and 55xc2x0 C., although higher temperatures, i.e. at least 35xc2x0 C., are often used when an optional alkanol is not included. Preferably, sterilization is carried out between 35xc2x0 C. and 45xc2x0 C. for a suitable increment of time; duration is shorter or longer dependent upon the concentration of coupling agent. Generally, the higher the concentration of coupling agent that is employed, the shorter will be the duration of treatment necessary to achieve effective sterilization; however, sterilization treatment is generally carried out for at least about 10 hours. Moreover, the inclusion of about 20 volume % of a lower alkanol can also somewhat shorten the needed duration of treatment. For example, in the absence of a lower alkanol, effective sterilization can be achieve; at a combination of concentration and duration equal to about 500-600 millimole hours. Thus, a treatment with about 25 millimoles of coupling agent for about 24 hours, or alternatively, treatment with 50 mM of coupling agent for about 12 hours, would meet this criterion. However, if for example 20% of isopropanol is included in the sterilization solution, treatment with 25 millimolar EDC for a period of even 3 hours, i.e. 75 millimole-hours, would be expected to achieve sterilization. Generally, the sterilization condition set forth in the aforementioned ""951 patent are believed to be equally applicable here, particularly from the standpoint of EDC concentration and duration of treatment. On the other hand, treatment for a period of about 24 hours is generally convenient for processing and is often used.
As indicated above, the sterilization treatment method is considered to be particularly useful for sterilizing allografts, such as a replacement ligament, tendon, bone, meniscus, vascular graft, heart valve or bioengineered tissue product having controlled resorption, as well as replacement organ components, such as heart valves, which have been made from animal tissue that has been suitably fixed so as to have desired physical properties. Prior to sterilization such material is first desirably rinsed with cold saline.
Subject to the considerations mentioned above, the biological tissue material being sterilized is usually maintained in contact with the sterilization solution for about 5 to 72 hours; the treatment is potently bactericidal, effectively inactivating even hard-to-kill bacteria and spores. However, because of the presence of the protecting agent, this sterilization treatment either does not significantly change the physical properties of the bioprosthetic tissue or changes it in a carefully controlled manner. Fresh tissue which has not previously been fixed, i.e. cross-linked, becomes only minimally, or alternatively controllably, cross-linked as a result of treatment, as can be seen by measurement of its shrinkage temperature or its resistance to enzymatic digestion, and thus either retains its desired physical properties or has them change somewhat in a carefully controlled manner. For example, when bioengineered tissue products are being treated, the resistance to cross-linking, as seen particularly during collagenase digestion testing, could be modulated to meet specific criteria an increase in shrinkage temperature of not more than about 2xc2x0 C. will usually be effected.
The present invention is further described by the examples that follow. These examples are not to be construed as limiting in any way either the spirit or the scope of the present invention.
Items which are to be implanted in the human body are required to be sterilized in a manner to effectively destroy all microorganisms. Due to the unique applications of liquid chemicals for use in sterilization processes, it is necessary to be vigilant in detecting, screening and testing microorganisms which could pose significant resistance to the sterilization process. Examples of reference microorganisms which have previously demonstrated high resistance to liquid chemical sterilants are: the spores of Bacillus subtilis, Clostridium sporogenes, Bacillus pumilus, Chaetonium globosom and Microascus cinereus, and representative vegetative cells, such as Mycobacterium chelonae, Methylbactrium extorquens and Trichosporon aquatile. Of the foregoing, the most resistant may be the spores of Bacillus subtilis. 
The preferred coupling agent that is used in the following examples is 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), which is commercially available. Peptone water is prepared by dissolving 1g of Bacto Peptone in 1 liter of de-ionized water, and the solution is then filtered into sterile bottles using sterile 0.2 micron filters. Coupling agents and/or protecting agents are solubilized in 10 mM HEPES buffer containing 0.85% of sodium chloride, pH 6.5 (HEPES buffer) or in 10 mM TRIS buffer containing 0.85% sodium chloride, pH 6.5 (TRIS buffer). Concentrations are expressed as mM (number of millimoles of chemical for each liter of solution), or as % (grams per 100 ml of solution). Temperatures are in xc2x0 C. (degrees Celsius), with room temperature being about 20-25xc2x0.
When porcine aortic roots that are fixed are employed, they are cross-linked according to the method described in U.S. Pat. No. 5,447,536 or glutaraldehyde-fixed or cross-linked or fixed with any other method used to preserve tissue. After fixation, the tissue is stored in 10 mM HEPES, 0.85% NaCl, 20% isopropyl alcohol, pH 7.4, at 4xc2x0 C. The sterility tests described in the following examples are conducted in the presence of bovine pericardium or porcine heart valve tissue. After such tissue is inoculated with microorganisms for test purposes and then submitted to sterilization, the solution is filtered through a 0.45 micron filter attached to a funnel (filter funnel). The tissue is washed for 20 minutes in a reciprocating shaker in the presence of peptone water containing Tween 80 in order to extract all indigenous spores or microorganisms from the tissue. This solution is then filtered through the same respective filter. The filters are then rinsed with peptone water to eliminate residual chemicals on the membrane that may prevent growth of the organisms tested. The membrane filters are incubated on solid agar TSA plates (Millipore) at about 32xc2x0 to 33xc2x0 C., e.g. 32.5xc2x0 C. All microbiological testing is performed in a biological laminar flow hood to prevent contamination. The shrinkage temperature tests and the resistance to collagenase digestion tests are conducted as described in the ""536 patent.